Probing DNA Structure with Fluorescent Nucleosides

Etheno-dA is a fluorescent nucleoside which is especially useful in observing the transition between DNA structural types. It is quite base labile and should be deprotected with ammonium hydroxide at room temperature for 24 hours. Alternatively, ultraMild chemistry can be used. 2-Aminopurine and AP-dC (G-Clamp) are also useful fluorescent nucleosides.

Pyrrolo-dC is a fluorescent deoxycytidine analog that is an ideal probe of DNA structure and dynamics.1,2 It base-pairs as a normal dC nucleotide. An oligo fully substituted with pyrrolo-dC has the same Tm as the control dC oligo with the same specificity for dG. Its small size does not perturb the structure of the DNA helix and it is well tolerated by a number of DNA and RNA polymerases. It is highly fluorescent and its excitation and emission are well to the red of most fluorescent nucleotide analogs, which eliminates or reduces background fluorescence from proteins. Pyrrolo-dCTP has potential uses in biological assay development.

By attaching pyrene or perylene to the 5 position of deoxyuridine through a triple bond, the fluorophore is electronically coupled to the deoxyuridine base. This electronic coupling of the base and the fluorophore makes the fluorescence sensitive to the base pairing of the dU portion of the molecule, allowing the discrimination between perfect and one base mismatched targets.

The tricyclic fluorescent nucleoside analogues, 1,3-diaza-2-oxophenothiazine, tC, and 1,3-diaza-2-oxophenoxazine, tCo, are deoxycytidine analogs that have been shown to base pair faithfully with dG with virtually no disruption of the normal duplex structure.3-5 This means that the stability of the DNA duplex is not compromised as compared to the control regardless of DNA sequence. The fluorescence quantum yield of tC is essentially unchanged between single stranded and double stranded DNA - 0.21 for single stranded DNA and 0.19 for duplex DNA. Also, the fluorescence characteristics of tC are not sensitive to neighboring base combinations. tCO has been shown to be the brightest fluorescent nucleoside analogue in duplex context reported so far and even retains the majority of its fluorescence when surrounded by guanine residues. Indeed, tCO has been reported to be 25-50 times brighter than 2-aminopurine.

The base analogue tCnitro is a FRET-acceptor together with tCO (or tC) as the donor molecule. This constitutes the first ever description of a nucleobase FRET-pair. This novel FRET-pair provides a unique tool for investigations of nucleic acid containing systems. tCnitro is virtually non-fluorescent under normal conditions.

References

1. D.A. Berry, et al., Tetrahedron Lett, 2004, 45, 2457-2461.

2. The Glen Report, 2007, 19, 8-9.

3. P. Sandin, et al., Nucleic Acids Res., 2008, 36, 157-167.

4. P. Sandin, et al., Nucleic Acids Res., 2005, 33, 5019-5025.

5. K.C. Engman, et al., Nucleic Acids Res., 2004, 32, 5087-5095.

7 Product Results
 
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  • Etheno-dA-CE Phosphoramidite
    Etheno-dA-CE Phosphoramidite (10-1006)
    Catalog #: 10-1006  |  CAS #: 160702-08-9
    Formal Name: 5'-Dimethoxytrityl-etheno-deoxyAdenosine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • Pyrrolo-dC-CE Phosphoramidite
    Pyrrolo-dC-CE Phosphoramidite (10-1017)
    Catalog #: 10-1017  |  CAS #: 644962-92-5
    Formal Name: 5'-Dimethoxytrityl-[6-methyl-pyrrolo-[2,3-d]-pyrimidine-2(3H)-one]-2'-deoxyribonucleoside, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • Pyrene-dU-CE Phosphoramidite
    Pyrene-dU-CE Phosphoramidite (10-1590)
    Catalog #: 10-1590
    Formal Name: 5'-Dimethoxytrityl-5-(pyren-1-yl-ethynyl)-2'-deoxyUridine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • Perylene-dU-CE Phosphoramidite
    Perylene-dU-CE Phosphoramidite (10-1591)
    Catalog #: 10-1591
    Formal Name: 5'-Dimethoxytrityl-5-(perylen-3-yl-ethynyl)-2'-deoxyUridine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • tC-CE Phosphoramidite
    tC-CE Phosphoramidite (10-1516)
    Catalog #: 10-1516
    Formal Name: 5'-O-(4,4'-Dimethoxytrityl)-1'-(1,3-diaza-2-oxophenothiazin-1-yl)-2'-deoxy-ß-D-ribofuranosyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • tC°-CE Phosphoramidite
    tC°-CE Phosphoramidite (10-1517)
    Catalog #: 10-1517
    Formal Name: 5'-O-(4,4'-Dimethoxytrityl)-1'-(1,3-diaza-2-oxophenoxazin-1-yl)-2'-deoxy-ß-D-ribofuranosyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
  • tCnitro-CE Phosphoramidite
    tCnitro-CE Phosphoramidite (10-1518)
    Catalog #: 10-1518
    Formal Name: 5'-O-(4,4'-Dimethoxytrityl)-1'-(7-nitro-1,3-diaza-2-oxophenothiazin-1-yl)-2'-deoxy-ß-D-ribofuranosyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
 
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